Effects of supplemental energy and protein on forage digestion and urea kinetics in beef cattle

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EFFECTS OF SUPPLEMENTAL ENERGY AND PROTEIN ON FORAGE DIGESTION AND UREA KINETICS IN BEEF CATTLE

by

ERIC ARTHUR BAILEY

B.S., West Texas A&M University, 2007

A THESIS

submitted in partial fulfillment of the requirements for the degree

MASTER OF SCIENCE

Department of Animal Sciences and Industry College of Agriculture

KANSAS STATE UNIVERSITY Manhattan, Kansas

2010

Approved by:

Major Professor Evan C. Titgemeyer

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Abstract

Two experiments quantified effects of supplemental protein and energy on forage digestion and urea kinetics in beef cattle. In experiment 1, energy treatments included: control, 600 g glucose dosed ruminally once daily, and 480 g VFA infused ruminally over 8 h daily. Casein was dosed ruminally once daily (120 or 240 g). Cattle (208 kg) had ad libitum access to low-quality hay (5.8% protein). Infusion of VFA decreased forage intake by 27%. Glucose decreased NDF digestibility. Microbial N flow was greater for 240 than for 120 g/d casein, but was not affected by energy. Retained N increased with casein supply. Urea-N entry rate (UER) and gut entry of urea-N (GER) were not affected by energy, casein, or interactions, but GER/UER was less when 240 rather than 120 g/d casein was provided. Compared to VFA, glucose tended to increase GER/UER. Glucose led to more microbial uptake of recycled urea than VFA. In these young calves, changes in N and energy supply did not greatly impact urea kinetics, likely because increased N was largely retained. In experiment 2, treatments included: 0 or 1.2 kg glucose, and 240 or 480 g casein. Cattle (391 kg) were fed low-quality hay (4.7% protein). Glucose reduced forage intake by 18%, whereas casein did not affect it, and depressed fiber digestion. Microbial N flow to the duodenum and retained N increased as casein increased, but neither was affected by glucose. Increasing casein increased UER 50%. Urinary urea-N increased as casein

increased; moreover, GER numerically increased 25% as casein increased. GER/UER decreased as casein increased. Glucose decreased urinary urea, but did not change UER or GER.

Microbial uptake of recycled urea was least for steers receiving 480 g/d casein with no glucose, reflecting that this treatment exceeded ruminal requirement for N. In these more mature steers, increases in N intake increased UER, reflecting that only small proportions of the increased N

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less N was retained. These studies demonstrate the influence of urea recycling in meeting N needs of cattle fed low-quality forage.

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List of Figures

Figure 1. The effect of ruminal energy (glucose or VFA) supplementation on ruminal pH in steers fed prairie hay ... 63 Figure 2. The effect of ruminal casein and energy (glucose or VFA) supplementation on ruminal ammonia concentration in steers fed prairie hay ... 64 Figure 3. The effect of ruminal energy (glucose or VFA) supplementation on ruminal acetate

concentration in steers fed prairie hay. ... 65 Figure 4. The effect of ruminal energy (glucose or VFA) supplementation on rumen propionate

concentration in steers fed prairie hay ... 66 Figure 5. The effect of ruminal energy (glucose or VFA) supplementation on rumen butyrate

concentration in steers fed prairie hay. ... 67 Figure 6. The effect of glucose supplementation on ruminal pH in steers fed prairie hay ... 98 Figure 7. The effect of casein and glucose supplementation on ruminal ammonia concentration in

steers fed prairie hay ... 99 Figure 8. The effect of glucose supplementation on ruminal acetate concentration in steers fed

prairie hay. Glucose was dosed ruminally once daily at feeding ... 100 Figure 9. The effect of glucose supplementation on ruminal propionate concentration in steers

fed prairie hay. ... 101 Figure 10. The effect of glucose supplementation on ruminal butyrate concentration in steers

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List of Tables

Table 1. Chemical composition of hay and supplements ... 57 Table 2. Effects of ruminal casein and energy (glucose or VFA) supplementation on intake,

digestion, and N balance of steers fed prairie hay ... 58 Table 3. Effect of ruminal casein and energy (glucose or VFA) on nutrient flows to the

duodenum and microbial efficiency in steers fed prairie hay ... 59 Table 4. Effect of ruminal casein and energy (glucose or VFA) supplementation on urea kinetics in steers fed prairie hay ... 60 Table 5. Effect of ruminal casein and energy (glucose or VFA) supplementation on plasma

metabolite concentrations and renal clearance in steers fed prairie hay ... 61 Table 6. Effect of ruminal casein and energy (glucose or VFA) supplementation on ruminal

fermentation characteristics in steers fed prairie hay ... 62 Table 7. Chemical composition of hay and supplements ... 92 Table 8. Effects of ruminal casein and glucose supplementation on intake, digestion and N

balance of steers fed prairie hay ... 93 Table 9. Effect of ruminal casein and glucose supplementation on nutrient flows to the

duodenum and microbial efficiency in steers fed prairie hay ... 94 Table 10. Effect of ruminal casein and glucose supplementation on urea kinetics in steers fed

prairie hay ... 95 Table 11. Effect of ruminal casein and glucose supplementation on plasma metabolite

concentrations in steers fed prairie hay ... 96 Table 12. Effect of ruminal casein and glucose supplementation on ruminal fermentation

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Acknowledgements

First off, I would like to acknowledge my major professor, Evan Titgemeyer. I really appreciate the opportunity he gave me when he allowed me to study under his tutelage here at Kansas State University. I have learned a great deal about not only my career but also about life from him, and for that I will be forever grateful. Cheryl Armendariz was instrumental in helping me get the lab aspects of my project completed. I had no previous experience working in a setting like that before, and her patience and willingness to help me means a lot to me. Hyatt Frobose, Mindy Fox, and Kristen Walker were my undergraduate student workers. They all did a great job and really went above and beyond the call of duty numerous times. Derek Brake is another student under Dr. Titgemeyer’s guidance. He helped with both the animal part and more importantly, when it came time to making urea recycling calculations. I couldn’t have done it without him. I would also like to thank the various KSU Ruminant Nutrition grad students who helped with my research projects and who helped me assimilate into life in Kansas. At first, I missed home terribly, but luckily I found a welcoming group of friends who helped me settle in and become comfortable in Manhattan. Finally, I would like to thank my family, my mom, dad, and little brother. I have missed them while I have been away at school and I want to thank them for their love and support throughout the entire process. Also, I cannot forget my good buddies Miles Zachary, J.W. Wagner, and Rodney Fuston. These guys are my three best friends in the world and their willingness to lend me an ear when I needed someone to talk to is something I am grateful for.

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CHAPTER 1 - A Review of Literature

Producers raising cattle on pasture have many production variables to consider.

Manipulating utilization of perennial pastures has proven to be a cost effective nutrition strategy for producers, because of relatively low costs associated with production of perennial forages. Low quality forage (CP<7%) can be detrimental to pasture utilization and animal performance. Cattle consuming a diet consisting only of low quality forages will not be able to achieve optimal performance due to a variety of limitations. Therefore, providing feed supplements is necessary to improve pasture utilization; however, costs associated with supplementation are large.

Due to the low quality of some pastures, producers are often forced to provide supplemental nutrients to their cattle. The question then becomes, “What nutrients must be provided to help maintain performance of the animals in question?” Researchers have evaluated many different answers to this question over the years, with varying levels of success.

Protein and energy have been provided to ruminants as means of improving performance. The benefits of providing supplemental protein to cattle consuming low quality forages are stark and well defined (DelCurto et al., 1990a; Köster et al., 1996). Protein has historically been the most expensive nutrient to use in production settings. This forced the scientific community to take a close look at protein supplements fed to ruminants.

The NRC (1996) classifies protein into two groups: degradable intake protein (DIP) and undegradable intake protein (UIP). DIP is protein degraded in the rumen by the resident microflora. Through DIP supplementation, ruminants may receive protein in the form of microbial cell protein. UIP escapes ruminal degradation, arriving in the small intestine of

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DIP Supplementation

Ruminant diets consisting of low-quality forage are most deficient in DIP (Köster et al., 1996). Peptides, amino acids, and ammonia are products of protein degradation in the rumen. Rumen microbes use the aforementioned nitrogenous products to support growth. A decrease in forage digestion occurs in times of inadequate ruminally available nitrogen (RAN) due to a decrease in microbial activity. This has a negative effect on the performance of the animal. With slower digestion, feedstuffs spend more time in the rumen. Concurrently, a reduction in intake occurs, reducing the amount of energy available to the animal.

Satter and Slyter (1974) were among the first researchers to show that increasing ammonia concentration in a fermentation system increased the productivity and growth of the microbial population. They found that 1.4 mM ammonia was sufficient to support maximal microbial cell protein production in the rumen, but they recommended 3.6 mM to provide a margin for safety.

Köster et al. (1996) provided definitive research on the efficacy of DIP supplementation in improving utilization of low-quality forage by cattle. They provided cannulated cattle (body weight [BW] = 588 kg) with increasing levels of sodium caseinate as a DIP supplement. This allowed the researchers to study the effect of DIP on forage intake and digestion without any confounding factors in the supplement, as had been a problem with previous research in this area. The provision of up to 540 g/d casein improved forage organic matter intake (OMI). Maximal total tract digestibility of neutral detergent fiber (NDF) was achieved at 180 g/d casein. Koster et al. (1996) recommended providing 11% of TDN as DIP to optimize total digestible OMI.

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consisted of three independent studies in which ruminally cannulated steers were fed one of four levels of supplemental DIP daily. The levels of supplementation were 0, 0.041, 0.082 and 0.124% of BW daily. Quality of forage differed among the experiments. The first trial used Bermudagrass, which had a CP content of 8.2%. The second trial used bromegrass with a CP content of 5.9%, and the third trial used forage sorghum with a CP concentration of 4.3%.

Supplementation of DIP had no effect on forage intake or digestion when the cattle were fed Bermudagrass. The only effects were increases in ruminal concentrations of ammonia and minor VFA (isobutyrate, valerate, and isovalerate). For the trial with bromegrass, there was a numeric increase in forage intake and total tract digestion of NDF in response to DIP

supplementation. Total digestible OMI was increased by DIP supplementation. Ammonia nitrogen concentrations in the rumen were increased with increasing DIP. Ammonia concentrations were lower at all levels of DIP supplementation in this trial than in the

Bermudagrass trial. Supplementation altered the ruminal VFA profile. The acetate:propionate ratio and butyrate concentration decreased with increasing DIP. Once again, the concentrations of the minor VFA were increased by protein supplementation.

The forage sorghum trial showed the largest effects of DIP supplementation. Forage intake and total tract digestibility of both OM and NDF were increased with DIP

supplementation, as was passage rate of acid detergent insoluble ash (ADIA). With poor-quality forage, rate of passage slows due to a slow rate of particle size reduction. Ammonia nitrogen and total VFA concentration increased with protein supplementation. The VFA profile showed the same changes as in the bromegrass trial.

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UIP Supplementation

Supplementation of protein sources high in UIP concentration is effective in improving forage utilization because of the ability of cattle to recycle nitrogen to the rumen. Bandyk et al. (2001) fed steers low quality forage and infused protein into either the rumen or the abomasum, allowing a comparison of the effects of DIP vs. UIP on forage intake and digestion. Providing a protein supplement improved forage intake, with the improvement being greater when DIP was provided. Organic matter digestibility increased with supplemental protein, with no difference between ruminal and postruminal infusions. Total tract digestibility of NDF was not improved by protein supplementation. Ruminal ammonia N level increased with protein supplementation, the magnitude of increase being larger for ruminal than for postruminal administration.

Wickersham et al. (2009) provided four levels of casein (0, 0.062, 0.124, or 0.186 g/kg BW per head per day) postruminally to steers consuming low quality forage. Urea kinetics were concurrently measured in the steers. They found that even at the highest level of UIP

supplementation (0.186 g/kg BW per head per day), steers still recycled 86% of all urea produced in the body back to the rumen. Forage OMI increased quadratically with larger

amounts of casein infused into the abomasum. Total tract digestibility of NDF was not impacted by infusing casein postruminally.

Effects of Energy Supplementation to Low-Quality Forage Diets

Low-quality forage can also be deficient in energy; therefore, attempts to improve animal performance have been made using supplemental energy. Supplemental energy has been

provided through various sources. Different sources of energy can have major effects on the productivity of cattle fed poor-quality forage, creating a need for research on various sources of

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Research has shown that supplementation of low-quality forage with higher-quality forages causes decreased intake of the low-quality forage. A substitution effect occurs in which low-quality forage consumption decreases with increasing high-quality forage provision. Costs associated with feeding high-quality forages are large; moreover, they take up more storage space than other supplements. There can also be problems in delivering the supplement, especially in production settings with difficult terrain.

Feed ingredients containing significant amounts of non-structural carbohydrates (NSC) have greater energy density than forages. This group includes corn grain, barley, and wheat middlings. Throughout the majority of the 20th century, prices of these grains were low, leading to an opportunity to provide inexpensive supplements. Conversely, research has repeatedly shown that large amounts of NSC supplementation can have negative impacts on forage intake and digestion.

Chase and Hibberd (1987) provided 0, 1, 2, or 3 kg/d of ground corn to mature cows. Total dry matter intake increased with the provision of supplements but the response did not match the magnitude of increase in supplement at each level. Consequently, forage OMI and forage DMI decreased linearly as corn supplementation increased. Digestion of NDF decreased cubically with supplementation. Ammonia nitrogen concentration decreased linearly also with provision of ground corn. This suggested that the starch-utilizing bacteria were capturing RAN to the exclusion of fiber-utilizing bacteria. Horn and McCollum (1987) discussed this effect in subsequent research. According to their theory, fiber digesting bacteria without adequate RAN were less productive, thus explaining the decreases in NDF digestion.

A “carbohydrate effect” also could partially explain the decrease in fiber digestion. Discussed by Arroquy et al. (2005), the carbohydrate effect was classified as a depression in

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fiber digestion by NSC supplementation independent of any pH effects. They stated that rumen microbes have an affinity for readily digestible sources of nutrients. When offered a greater vs. a less digestible source of nutrients, the microbes acted upon the more digestible source of

nutrients more readily.

Ruminal pH can have a negative impact forage digestion. Rapid digestion of grains occurs in the rumen, leading to swift decreases in ruminal pH. A pH below 6.2 can inhibit the productivity of cellulolytic bacteria (Mould et al., 1983); however, for a number of trials that evaluated energy supplementation in low-quality forage diets (Olson et al., 1999; Krysl et al., 1989; Pordomingo et al., 1991), pH never strayed below the 6.2 threshold.

A number of researchers have tried to ameliorate the depressing effects of low RAN when supplementing NSC. DelCurto et al. (1990) fed ruminally-cannulated steers two levels of supplemental protein and two levels of energy within each level of protein. At the low level of supplemental protein, provision of the high level of energy (18.4 kcal ME/kg BW daily)

depressed forage intake and NDF digestion. Overall dry matter (DM) digestion increased when readily digestible carbohydrates were fed. When provided the high level of supplemental

protein, the negative effects of energy supplementation were ameliorated. No differences existed between the two levels of energy supplementation within the high protein level, but forage intake improved numerically at the high energy level. Even with a small change in energy level, NDF digestibility still decreased.

Olson et al. (1999) extended this line of research by evaluating two levels of starch supplementation and four levels of protein within each level of starch. Animals received a low quality forage diet (4.9% CP). Starch feeding levels were 0, 0.15, or 0.30% of BW per day. Levels of protein supplementation were 0, 0.03, 0.06, 0.09, or 0.12% of BW per day. Within

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each level of starch, forage DM and NDF intakes increased with increasing supplemental protein. With increasing levels of starch, forage DM and NDF intakes were depressed.

Increasing starch depressed total tract digestion of NDF. Within each level of starch, increasing protein increased NDF digestion. Not even the highest level of protein (0.12% of BW/d) within each level of starch returned NDF digestion to levels similar to protein supplementation alone. Total tract digestion of DM and OM were most affected at the high level of starch

supplementation. At the low level of starch, increasing levels of supplemental protein did not improve digestion to the levels observed for the control animals.

Klevesahl et al. (2003) studied the effects of a wide range of protein levels on forage intake and digestion when supplementing 0.30% of BW/d of starch to a forage-based diet. Fourteen ruminally-cannulated steers were used in a two-period, 14-treatment study. Animals were given one of seven levels of DIP (0, 0.015, 0.051, 0.087, 0.123, 0.159, or 0.195% of BW DIP per day) and received starch in either period one or two. The diet consisted of grass hay (4.9% CP). Intraruminal dosing of DIP occurred once daily for all steers.

Both the DIP and the starch had independent effects on forage OM and NDF intake; there were no significant DIP x starch interactions for intake. Starch depressed intake, whereas DIP yielded a quadratic response. Up to 0.123% of BW/d of supplemental DIP increased intake of forage OM and NDF, but intakes were decreased relative to maximum at levels above this. A significant DIP x starch interaction occurred for total tract NDF digestion. With no supplemental DIP and 0.30% of BW/d starch, NDF digestion was ~30%. As level of DIP increased, so did NDF digestion, up to ~50%, the level observed with DIP supplementation only. Starch without any supplemental DIP resulted in a depression of total tract NDF digestion by 20 percentage

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units. Supplemental DIP increased NDF digestion by approximately 5 percentage units in the animals that did not receive starch.

Supplemental starch decreased rumen pH, but the lowest pH was 6.33, greater than the proposed pH of 6.2 for inhibition of cellulolytic bacteria (Mould et al., 1983). Total VFA and ammonia concentrations increased linearly with increasing DIP. The VFA profile exhibited some interesting differences. Acetate concentration decreased with the inclusion of starch in the diet. Concurrently, concentration of propionate and butyrate increased. Supplemental DIP linearly increased the proportions of isobutyrate, valerate, and isovalerate.

In the aforementioned studies, starch was the source of supplemental energy. Other research has evaluated the effects of different types of NSC on forage intake and digestion. Heldt et al. (1999b) looked at this effect through a series of trials. The first trial utilized 13 ruminally cannulated steers with a 2 x 3 x 2 factorial treatment design. This included two levels of DIP (0.031 and 0.122% of BW daily) and three distinct carbohydrate sources (starch, glucose, and fiber) that were fed at two levels (0.15 and 0.30% of BW daily). Another set of steers were fed only hay to provide a baseline measurement of forage intake and digestion. Animals received their supplements once daily intraruminally.

Starch, glucose, and fiber each exhibited unique effects. Starch and DIP did not interact. The level of DIP supplemented did not affect forage OMI. However, NDF digestibility differed by approximately 6% when comparing high and low DIP supplementation. The cattle receiving high DIP treatment digested a greater amount of NDF than those receiving the low DIP

treatment.

At the low level of DIP, 0.15% of BW daily as glucose supplementation did not affect forage OMI intake or NDF digestibility, but the 0.30% of BW daily level of glucose depressed

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forage OMI and NDF digestion to levels below that of the negative control cattle that only received forage. However, at the high level of DIP, forage OMI and NDF digestion were

significantly improved by both levels of glucose supplementation. This once again suggests that glucose-related depressions in ruminal fermentation are related to a deficit in RAN, because an increase in the supplementation of DIP ameliorated the depression associated with glucose supplementation.

The addition of supplemental fiber produced more variable results. At the low level of DIP supplementation, the low level of fiber increased forage OMI above that of the negative control, but the high level of fiber depressed forage OMI to a level below the negative control. The NDF digestibility at both levels of fiber supplementation was greater than when glucose was supplemented. At the high level of DIP, fiber supplementation improved forage OMI intake to a level above that observed for starch supplementation, but below that observed for glucose supplementation. NDF digestibility did not differ between supplemental glucose and

supplemental fiber at the high level of DIP supplementation. NDF digestion improved when comparing the high fiber to the low fiber within the high level of DIP. This effect did not occur at the low level of DIP. Perhaps RAN was not adequate to optimize microbial function and thus depressed NDF digestibility. It is somewhat difficult to interpret NDF digestibility in response to fiber supplementation due to the fact that both the forage and supplement contribute to overall dietary NDF. NDF digestibility would be confounded in cases where the quality of the fiber supplemented was much higher than that of the basal forage in the diet. Improvements in basal forage utilization in response to fiber supplementation have been seen in other research (Highfill et al., 1987; Martin and Hibberd, 1990).

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The varied sources of supplemental carbohydrates in the work of Heldt et al. (1999b) impacted ruminal fermentation characteristics. Rumen pH generally decreased with the

inclusion of supplemental carbohydrates. The higher level of carbohydrate inclusion magnified this effect. Rumen ammonia levels were well below the recommendations of Satter and Slyter (1974) when animals were given the low DIP treatment. Conversely, the high DIP treatment improved ruminal ammonia concentrations. Ruminal ammonia concentrations were less for the glucose supplements than for starch and fiber treatments at the high DIP level. Possibly, the provision of supplemental glucose led to a greater uptake of rumen ammonia than the other treatments. This would make adequate DIP supplementation necessary to prevent depressions in forage intake and digestion with provision of supplemental glucose.

The VFA profile of the cattle showed differences among treatments. Across the board, supplemental DIP and supplemental energy decreased the proportion of acetate. Increasing the amount of DIP or energy magnified this effect. Generally, supplementation increased the proportion of propionate. Due to butyrate and acetate having the same precursor (acetyl CoA), any reductions in acetate concentration were followed by a concurrent increase in butyrate concentration. Glucose supplementation increased butyrate concentration more than the other energy treatments. Lactate concentration was increased by glucose treatments. Supplementation in general and increasing the level of DIP increased the molar proportions of isobutyrate,

isovalerate, and valerate. Studies previously mentioned in this review (Köster et al., 1996; Olson et al., 1999) showed that DIP supplementation increased the proportion of the three minor VFA. Heldt et al. (1999b) noted in their discussion that they believed the impact of DIP to be more important than the impact of carbohydrate supplementation on the proportions of minor VFA.

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Fermentation of certain amino acids produces branched chain organic acids that are growth factors for fibrolytic bacteria (Baldwin and Allison, 1983).

In a series of trials conducted by Heldt et al. (1999a), the effects of various supplemental sugars and starch fed to steers in combination with DIP were evaluated. The first trial paired an insufficient level of DIP (0.031% of BW/d) with one of four carbohydrate sources (starch, glucose, fructose, or sucrose) fed at 0.30% of BW/d. Level of supplemental DIP was increased to 0.122% of BW/d in the second trial.

In the first trial, no differences were found between starch vs. sugar, monosaccharide vs. disaccharide, or glucose vs. fructose when evaluating intake and digestion parameters (forage OMI, digestible OMI, OM digestibility, and NDF digestibility). Conversely, important

differences were found when comparing all supplemented animals to the negative control. The supplemented animals all had depressions in NDF digestibility when compared to the negative control and forage OMI numerically increased with supplementation.

The greater level of DIP supplemented in the second trial led to different effects of carbohydrates on forage intake and digestion. Forage OMI increased when supplements were provided. No differences existed among carbohydrate treatments with regard to forage OMI. Organic matter digestibility also increased with supplementation. Supplemental starch led to lower OM digestibility than did supplemental sugar. Glucose and fructose supplementation led to higher OM digestibility than did sucrose supplementation. In a subsequent comparison, no differences were observed between glucose and fructose. The effects on OM digestibility were similar to those on NDF digestibility, suggesting that differences in OM digestibility among treatments were due to differences in NDF digestion. Supplementation of glucose and fructose led to much higher NDF digestion than did starch when the greater level of DIP supplement was

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provided. This provided additional evidence that the shortcomings of simple sugar

supplementation can be overcome with additional RAN, whereas the mechanism by which starch inhibits forage digestibility may warrant further investigation.

The effects of supplemental carbohydrates on the VFA profile of the steers were similar for both experiments. There were numeric differences, but the overall trends were the same, suggesting that effects were the result of the energy supplements. It also suggested that increasing DIP did not further alter the VFA profile. Throughout both experiments,

supplementation resulted in a decrease in the molar proportion of acetate, with the decrease being larger when sugars were provided. Supplementation increased the proportion of propionate, with no differences among the energy treatments. When acetate concentration decreased, butyrate concentration increased; this effect manifested itself with sugar supplements more than with starch supplements. Results for the minor VFA (isobutyrate, valerate, and isovalerate) were mixed. Starch supplementation increased the proportion of all three minor VFA; however, the increase of isobutyrate and isovalerate were greater for starch than for any of the sugars. These somewhat unexpected results were possibly due to the sugars being

supplemented in conjunction with DIP. Valerate increased significantly with provision of supplemental sugar. There were differences between starch vs. sugar and also for

monosaccharide vs. disaccharide. Sugar supplementation also increased the concentration of lactate to levels much greater than those of the control or the starch treatment.

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Protein to Energy Ratio

A number of researchers have proposed the concept of an ideal energy (total digestible nutrients; TDN) to protein ratio in the rumen. This approach considers the digestibility of the forage, instead of just the crude protein concentration of the forage, with the hope of finding an ideal ratio of protein to energy for maximizing efficiency of ruminal microbes. Moore et al. (1999) evaluated this ratio in a review article. Their database included 444 comparisons between unsupplemented controls and supplemented treatments. The database included various sources of forage and supplements. Forages were grouped into cool season, warm season, native grass, and straw. Supplements included high protein supplements, high energy supplements, liquid feeds, supplements containing non-protein nitrogen, by-product feeds, and plant supplements.

Supplementation had both positive and negative effects on voluntary feed intake. Intake of forages with a TDN:CP ratio of less than 7:1 generally decreased in response to

supplementation, whereas forages with a TDN:CP ratio above 7:1 generally had the opposite effect. A TDN:CP ratio of less than 7:1 indicates a relatively high concentration of protein in the forage in relation to the amount of energy available. Hence, the observation that

supplementation decreases voluntary intake when the TDN:CP ratio decreases below 7:1 might be expected because the protein concentration should be nearly sufficient to support the rumen ecosystem.

Moore et al. (1999) further investigated the relationship of TDN:CP ratio by exploring the effect of supplemental TDN intake on the change in voluntary forage OMI, as classified by the forage TDN:CP ratio. Generally, when forage TDN:CP ratio was less than 7:1, supplemental TDN intake almost always decreased voluntary OMI. Results for observations with a TDN:CP ratio above 7:1 were mixed. The observations were then sorted by feed type (protein, energy,

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supplemental TDN intake was greater than 0.7% of BW daily, voluntary forage intake always decreased.

In summary of the review by Moore et al. (1999), supplements provided to a forage with a TDN:CP ratio of greater than 7 increased forage intake. Also, providing supplemental TDN at a level above 0.7% of BW daily decreased voluntary forage intake.

Bowman et al. (2004) explored the TDN:CP ratio in grazing cows. The first portion of their research consisted of a digestion trial utilizing cross-bred heifers fitted with ruminal cannulae. The second half of their research involved pregnant crossbred cows in a two-year grazing trial. In both trials, researchers fed increasing levels of NSC and measured effects on forage intake and digestion. Supplements in both trials were fed at three levels (0.32, 0.64, or 0.96 kg/d) with TDN:CP ratios of 9.7, 9.5, and 9.7 for the 0.32, 0.64, and 0.96 kg levels of supplementation, respectively. The diets were not iso-nitrogenous but they were designed to provide 0.34 kg/d of CP and 5.1 Mcal of ME for both trials.

The heifers in the first trial were given ad-libitum access to low-quality orchardgrass (5.5% CP) that had a TDN:CP ratio of 10:1. The cows in the second trial grazed native rangeland with a TDN:CP ratio of 7:1 in the fall after weaning. It is important to note that the heifers used in the first trial received forage considered to be deficient in protein based on the TDN:CP ratio of Moore et al. (1999), whereas the cows grazing native range had access to forage considered adequate in protein (forage TDN:CP < 7). In the second portion of the trial, two ruminally-cannulated cows were included with each treatment to facilitate the collection of forage extrusa samples via ruminal evacuation.

For the first experiment, intake of forage and total dry matter, organic matter, NDF, and CP increased with provision of supplements. There were no differences among the levels of

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supplemental NSC. Total tract digestion of forage and total diet DM, OM, and NDF increased with supplementation; however, there were quadratic effects of treatments for forage DM

digestion, forage OM digestion, and forage NDF digestion. Forage OMI increased up to the 0.64 kg/d-level of supplementation, along with an improvement in forage NDF digestion. At 0.96 kg/d (the only level above 0.64 kg/d), intake of forage DM and forage OM were decreased, along with a concurrent decrease in forage NDF digestion. Improvement in low-quality forage

utilization when high-fiber supplements were provided to beef cattle was previously demonstrated (Heldt et al., 1999a; Highfill et al., 1987; Martin and Hibberd, 1990).

As previously mentioned, the cattle used in the grazing trial of Bowman et al. (2004) had access to forage considered adequate in protein based on the forage TDN:CP ratio. Collection of the ruminal extrusa from cows in each treatment group yielded no significant differences in composition. There was variation in forage quality between the two years (5.1% CP in year one vs. 6.2% CP in year two) but there were no interactions between year and treatments.

For the first year of the grazing trial, there were distinct linear effects of NSC level on forage and dietary intake measurements. Increasing supplemental NSC decreased intake of forage and total diet DM, OM, NDF, and CP. Interestingly, at the highest level of NSC supplementation, the dietary intake of CP was still greater than that of the non-supplemented control, yet intakes of DM, OM, and NDF were decreased, suggesting that NSC supplementation was having a deleterious effect on forage intake.

As previously mentioned, forage quality was greater in year two of the trial than in year one. Increasing the amount of supplemental NSC linearly decreased both forage and total dietary intake of DM, OM, NDF, and CP. Intake of forage OM the cows that received 0.96 kg/d of NSC decreased by 68% relative to the unsupplemented control. The authors attributed the

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decrease in forage utilization to the supplements, postulating that the TDN:CP ratio of the supplements was sufficiently high to create a imbalance in the energy to protein ratio in the rumen. This, in turn, would have limited the effectiveness of the ruminal microbial population.

Effect of VFA Infusions on Rumen Function

Ruminal fermentation produces VFA as products. Ruminant nutritionists have known about the role of VFA in providing energy to ruminants for over 50 years. The major VFA of interest in ruminant nutrition are acetate, propionate, and butyrate. VFA are the primary forms of energy ruminant animals receive from their symbiotic partners. Many feedstuffs are degraded to some extent in the rumen, meaning that the fundamental composition of feeds changes

between intake and absorption. A large body of research exists on the many facets of VFA production and utilization.

Early work in this area centered on the roles of VFA on the regulation of feed intake in ruminants. Montgomery et al. (1963) infused either acetic (870 g/d), propionic (280 g/d), butyric (260 g/d), or lactic acid (340 g/d) into the rumen of dairy cows. The acids were diluted with 4 liters of water and infused over 4 hours daily. Cows were fed alfalfa-bromegrass hay. Hay intake was decreased by infusions of acetic acid (35% decrease) and butyric acid (17% decrease). Ruminal pH was not affected by treatment; the lowest pH recorded in their

measurements was 6.5. Blood metabolites were measured in an attempt to identify a marker for intake inhibition. Increases in blood ketone and decreases in blood urea concentrations occurred in response to acetic acid infusion.

Simkins et al. (1965) also studied the effect of VFA infusions on feed intake by cattle. Their research used cattle fed either pelleted or chopped alfalfa. Isocaloric amounts of acetic

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15% butyrate) were infused intraruminally. The infusions were balanced to provide 15% of each animal’s daily digestible energy requirement. This led to differences in the actual amounts infused into the cows. Treatments were infused over a 5-hour period for three days. For the cows consuming alfalfa pellets, acetate infusion caused a greater decrease (-30%) in intake relative to other infusions. There were no differences in forage intake when cattle consuming chopped alfalfa were provided the same VFA infusions.

Simkins et al. (1965) also measured blood metabolites in hope of finding an explanation for the decreases in intake. Blood sugar, ketone, VFA, acetate, propionate, and butyrate

concentrations were measured from a jugular blood sample taken from each animal. Infusion of butyrate increased ketone and decreased sugar concentrations in the blood. Propionate infusion increased blood sugar concentrations. The VFA mixture decreased ketone concentrations. Based on the inconsistent effects of VFA infusion on the animal intakes and metabolite concentrations, Simkins et al. (1965) were unable to propose a suitable mechanism by which VFA infusions decreased intake of the basal diet.

Infusion of VFA above normal physiological levels may be the explanation for the decreases in intake reported in early research. Papas and Hatfield (1978) conducted a series of experiments to investigate the role of VFA infusions on decreases in feed intake. VFA were administered into the abomasum of sheep in a variety of concentrations. In their first experiment, six treatments were used: 1) water, 2) 60 g sodium acetate, 3) 40 g sodium

propionate, 4) 44 g acetic acid, 5) 31 g propionic acid, and 6) 25.8 g butyric acid. VFA in acid form decreased intake, whereas VFA salts had no effect on intake. This led the authors to blame the molar amounts of acid infused for depression of intake. To prove this, the authors conducted another trial where each animal received 0.5 mole (diluted to 750 mL with water) of acetate,

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propionate, butyrate, or hydrochloric acid per day. All treatments decreased intake relative to control, but urine pH did not decrease. In a subsequent trial, the animals received twice the molar amount of VFA as in the initial trial. The lambs receiving acid reduced their feed intake to less than 100 grams per day and developed metabolic acidosis. The authors concluded that mass production (or infusion) of VFA into the rumen upsets the acid-base balance of the body, leading to a number of systemic issues.

As observed in previous research, VFA can be provided to animals in one of two forms; in acid form or as sodium salts. Each method has its own challenges. In the acid form, a large dose of VFA will decrease rumen pH and potentially may cause a litany of digestive issues. The VFA salts will increase rumen osmolality. Increased osmolality by the addition of NaCl has been shown to decrease VFA absorption from the rumen (Lopez et al., 1994). Normally, osmotic pressure is lower in the rumen than in the blood, which allows for water to be absorbed from the rumen. If the osmotic pressure in the rumen rises above that of the blood, then water will be moved from the blood into the rumen.

Lopez et al. (2003) studied the effects of VFA supply on VFA absorption and on water kinetics in sheep maintained by intragastric infusions. Sheep were given one of three VFA infusion rates intraruminally. VFA, buffer, and macro minerals were infused ruminally while casein was infused into the abomasum. On data collection days, water and casein were withheld from the animals and VFA were infused at elevated levels (0.5, 1.4 and 1.8 times the basal infusion) within the solution. A marker for the ruminal liquid phase and total ruminal volume were used. Absorption of VFA from the rumen increased as the concentration of VFA in the infusion increased. Absorption from the rumen was less than the rate of infusion, such that VFA

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accumulated. This illustrated the risk associated with cattle consuming large meals of carbohydrates that can be rapidly fermented to VFA.

Intraruminal infusions of VFA that are within the physiological range produced by common diets and intake levels can still limit intake. Propionate, infused within the

physiological production range, consistently decreases feed intake by cattle. Diets rich in starch increase propionate concentration in the rumen. Ruminal propionate infusion decreased feed intake in dairy cows (Oba and Allen, 2003). In each of two experiments, they infused 8 different mixtures of acetate and propionate into the rumen of lactating dairy cows. The free-acid form of VFA were used in the first experiment, whereas VFA salts were used in the second experiment. Dry matter intake decreased with increasing proportion of infused propionate during both experiments. Total diet metabolizable energy (ME) intake also decreased with increasing infusion of propionate, in spite of the fact that ME content of the infusate increased with as concentrations of propionate in the infusate increased. This suggested that propionate did not play a role in regulating feed intake by cattle. Previous researchers suggested that the hypophagic effects of propionate were due to relatively high energy yielded though oxidation of propionate compared to other VFA.

Causing an aversion to the diet can be another possible mechanism by which VFA decrease voluntary feed intake. Research conducted by Ralphs et al. (1995) showed that gavage of animals with glucose or VFA had different effects on diet preferences. Each morning, sheep were offered straw with one of two unique flavors for 15 minutes. Animals were then ruminally gavaged with 200 mL of either a glucose or propionate solution which provided 13% of the daily energy requirement for the first experiment and 26% in the second experiment. After the

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gavage, animals were allowed access to feed for 45 minutes. A reduction in intake during the 45-minute feeding period was judged to indicate satiety.

For both experiments, glucose had no effect on hay intake. Propionate decreased intake in both experiments, with reductions being greater when the sheep received the larger dose of propionate. Propionate also caused aversions to both flavors of straw. For the last two days of each period, animals were offered a choice between both flavors. In the prior days of each period, only one flavor was given in combination with energy treatments. Animals always avoided the flavor that was associated with propionate, leading the authors to conclude that the propionate treatment in their second experiment caused a negative post-ingestive consequence in the animals due.

Villalba and Provenza (1997) investigated the role of VFA in feed preferences. They conducted four experiments to determine if different levels of VFA would cause the animals to develop preferences for or aversions to flavored feeds. In the first experiment, sheep were given one of four doses of sodium propionate (0, 4, 8, or 12% of daily DE requirement). Sheep used in the second experiment received one of four doses of sodium acetate at the same levels of daily DE requirement. Provision of sodium chloride intraruminally tested the effect of increased osmolality on intake in a third experiment. In the fourth trial, various combinations of acetate and propionate were administered to the sheep to discern preferences for either VFA.

In the first trial, intake of straw was depressed with provision of propionate at 8 and 12 percent of daily DE requirement. There were no differences in intake between the control and 4% level. In contrast, acetate did not have an effect on intake at gavage rates of 4 or 8% of DE but the 12% gavage rate of acetate depressed intake. Gavaging the sheep with only NaCl did not

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have any effect on intake of straw. Animals given VFA in the fourth trial increased intake compared to those given either water or NaCl.

A reduction in intake is not the only response to ruminal infusion VFA. Increasing VFA concentration in the rumen is associated with increased permeability to urea. Houpt and Houpt (1968) studied the transfer of urea-N across the ruminal wall. Using Pavlov pouches and two-balloon catheters, various concentrations of urea were injected into the jugular vein. The pouches contained fluid of varied ruminal ammonia concentrations. The pouches were treated with anti-microbial agents to inhibit urease activity. With the inhibition of urease, transfer of urea across the rumen wall related directly to the concentration difference between blood urea and rumen fluid. Without urease inhibition, urea entering the Pavlov pouch quickly was hydrolyzed to ammonia. Hydrolysis of urea to ammonia resulted in an increase of transfer of urea from the blood to the rumen. Kennedy and Milligan (1978) corroborated these findings.

Remond et al. (1993) measured the net transfer of urea and ammonia across the ruminal wall of sheep. They were interested in correlating blood flow and transfer of urea into the rumen. Sheep were fed a constant amount of orchardgrass hay daily. Animals received pulse doses of acetohydroxamic acid (a known urease inhibitor), butyric acid, ammonia, or sodium chloride during the end of each period of the trial. Carbon dioxide bubbled into the rumen was an additional treatment to mimic increased gas production. Ammonia absorption and blood flow to the rumen were measured, with correlations made between the two measurements. Ammonia absorption from the rumen increased with butyrate and CO2 treatments.

Acetohydroxamic acid decreased urease production in the rumen, leading to a decrease in rumen ammonia concentration. Intraruminal ammonia injection increased the net transfer of ammonia but had no effect on blood flow to the rumen. Conversely, NaCl increased ruminal

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osmolality and blood flow to the rumen. Concurrently, the net transfer of ammonia was

decreased. Carbon dioxide gas decreased concentration of VFA in the rumen. Net absorption of ammonia and blood flow to the rumen increased over time when the CO2 treatment was applied but the net transfer of urea decreased, leading the authors to postulate that blood flow is not related to net urea of transfer to the rumen, thus corroborating the findings of Dobson et al. (1971).

As rumen microbial activity increases, uptake of nitrogen by microbes in the rumen increases, allowing the digestibility of a diet to have an impact on urea kinetics in cattle. Increased VFA production and increased VFA concentrations in the rumen correspond with increased permeability of the ruminal epithelium to urea.

Kennedy (1980) investigated the effects of sucrose supplementation on the degradation of urea in cattle. Cattle were fed alfalfa hay with sucrose supplemented at either 0.5 or 1.0 kg/d. Urea kinetics were determined by intravenous infusion of 14C-urea. Provision of sucrose in the diet increased urea entry into the rumen by 35%. Production of microbial N improved with provision of sucrose, leading to lesser ruminal ammonia concentrations in those steers.

Norton et al. (1982) measured urea synthesis and degradation in sheep fed grass hay pellets and supplemented with flaked barley. Sheep were fed either 1 kg of pelleted grass cubes or 0.7 kg of pelleted grass cubes and 0.3 kg of flaked barley daily. Urea kinetics were measured with intravenous infusion of 14C-urea. Flaked barley supplementation increased the

concentration of butyrate in the rumen but had no effect on overall VFA concentration. The amount of recycled urea utilized in the rumen increased with provision of barley. Flow of urea into the rumen increased also among sheep fed flaked barley.

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Conclusion

Maximum performance and efficiency in the rumen cannot be achieved without the supplementation of protein to low-quality diets. The provision of supplemental energy can have detrimental effects on forage digestion without adequate provision of protein. Supplementation with readily digestible carbohydrates or VFA infusions will have a number of effects on ruminal function and nitrogen metabolism in ruminants. The mechanisms by which these effects are mediated require further study.

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Kennedy, P. M. 1980. The effects of dietary sucrose and the concentrations of plasma urea and rumen ammonia on the degradation of urea in the gastrointestinal tract of cattle. Br. J. Nutr. 43:125-140.

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Olson, K. C., R. C. Cochran, T. J. Jones, E. S. Vanzant, E. C. Titgemeyer, and D. E. Johnson. 1999. Effects of ruminal administration of supplemental degradable intake protein and starch on utilization of low-quality warm season grass hay by beef steers. J. Anim. Sci. 77:1016-1025.

Papas, A., and E. E. Hatfield. 1978. Effect of oral or abomasal administration of volatile fatty acids on voluntary feed intake in growing lambs. J. Anim. Sci. 46:288-296.

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Villalba, J. J., and F. D. Provenza. 1997. Preference for flavored wheat straw by lambs

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Wickersham, T. A., E. C. Titgemeyer, R. C. Cochran, and E. E. Wickersham. 2009. Effect of undegradable intake protein supplementation on urea kinetics and microbial use of recycled urea in steers consuming low-quality forage. Br. J. Nutr. 101:225-232.

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CHAPTER 2 - EFFECTS OF SUPPLEMENTAL ENERGY AND

PROTEIN ON FORAGE DIGESTION AND UREA KINETICS IN

GROWING BEEF CATTLE

1

E. A. Bailey, E. C. Titgemeyer, K. C. Olson,

D. W. Brake, M. L. Jones, and D. E. Anderson

1

This project was supported by National Research Initiative Competitive Grant no. 2007-35206-17848 from the USDA National Institute of Food and Agriculture.

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Abstract

We quantified effects of supplemental energy from differing sources on nutrient digestibility and urea kinetics at 2 levels of degradable intake protein. The study was a 6 × 6 Latin square with treatments arranged as a 3 × 2 factorial. Energy treatments included: control, 600 g glucose dosed ruminally once daily, and 480 g VFA (192 g acetic acid, 144 g propionic acid, 144 g butyric acid) infused ruminally over 8 h daily. Casein (120 or 240 g) was dosed ruminally once daily. Six ruminally- and duodenally-cannulated steers (208 kg) had ad libitum access to prairie hay (5.8% CP). We infused 15N15N-urea intravenously to measure urea kinetics. Infusion of VFA decreased (P < 0.01) forage intake by 27%; decreases in forage intake due to glucose (7%) and increases due to increasing casein (4.5%) were not significant. Dosing glucose decreased total tract NDF digestibility (P < 0.01) and tended to decrease ruminal NDF

digestibility; depressions in response to glucose tended to be greater at the lower level of casein. Increasing casein decreased ruminal pH (P < 0.02). Infusion of VFA decreased pH during the infusions, but not at other times, whereas glucose decreased pH 2 h after dosing. Ruminal concentrations of NH3, acetate, and propionate decreased, whereas those of butyrate increased, when glucose was supplemented; glucose may have exacerbated a ruminal NH3 deficiency. Increasing casein increased (P < 0.01) ruminal concentrations of NH3, acetate, propionate, isobutyrate, and isovalerate. Supplemental energy decreased plasma urea-N concentration (P = 0.03), whereas casein level did not affect it (P = 0.16). Microbial N flow was greater (P < 0.04) for 240 g/d than for 120 g/d casein but it was not affected by supplemental energy (P = 0.23). Urea-N entry rate (UER) and gut entry of urea-N (GER) were not affected (P ≥ 0.12) by supplemental energy or casein, but the proportion of UER that was recycled to the gut was less when 240 g/d rather than 120 g/d casein was provided (P = 0.01). When compared to VFA,

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= 0.01) microbial uptake of recycled urea than VFA. The lack of treatment effects on urea production, particularly in response to increased N supplied as casein, may reflect that the complete diets never provided excessive amounts of N and that increased provisions of intestinally-available AA were used efficiently by cattle for protein deposition.

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Introduction

Urea recycling is important to cattle grazing forages deficient in protein (CP < 7%). Wickersham et al. (2008) established that even when cattle consuming low-quality forage received adequate protein, they recycled large proportions (~95%) of urea production to the gastrointestinal tract. Supplementing cattle consuming low-quality forage with non-structural carbohydrates (NSC) is a strategy to increase energy intake by cattle but NSC supplementation without adequate protein content in the diet has detrimental effects on forage utilization (Heldt et al., 1999; Olson et al., 1999; Klevesahl et al., 2003). In contrast, NSC supplementation

reportedly increases microbial capture of recycled urea-N (Kennedy, 1980). Therefore, we wanted to investigate how differing sources of supplemental energy (to the animal vs. to the ruminal microbes) affected urea kinetics, forage intake, forage digestion, and the efficiency of N capture by ruminal microbes. Ruminal glucose should stimulate microbial growth, leading to an increase in the microbial cell protein supply to the animal. Conversely, no increases in the protein supply to the animal is expected when VFA are provided ruminally. Infusion of VFA has been associated with increased permeability of the rumen wall to urea (Norton et al., 1982); it has also been shown to increase blood flow to the rumen wall (Sellers et al., 1964).

Our hypothesis was that NSC supplementation would increase microbial growth and exacerbate a ruminal N deficiency, which would increase the amount of urea recycled to the rumen. In addition, we hypothesized that adding VFA to the rumen would increase the amount of urea recycled to the rumen by increasing the permeability of the rumen epithelium to urea. For this work, we supplemented protein at 2 levels, an amount observed to maximize forage intake and digestion (240 g/d; Heldt et al., 1999) and a deficient amount (120 g/d), to measure the effects of adequate and inadequate dietary N on urea kinetics.

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Materials and Methods

All procedures involving the use of animals were approved by the Institutional Animal Care and Use Committee at Kansas State University.

We studied the effects of providing supplemental protein and energy to growing beef steers consuming low-quality forage, with emphasis on urea kinetics. Six ruminally- and duodenally-cannulated Angus steers (average initial BW = 208 ± 17 kg) were used in a 6 × 6 Latin square with dietary treatments arranged as a 2 × 3 factorial. One of 2 protein treatments (120 or 240 g of sodium caseinate, New Zealand Milk Products Inc., Auckland, New Zealand; Table 1) were pulse dosed into the rumen once daily at 0630 h. One of 3 energy treatments were superimposed on protein treatments: 1) no supplemental energy (control), 2) 600 g glucose (dextrose monohydrate, ADM Corn Processing, Decatur, IL; Table 1) pulse-dosed into the rumen once daily at 0630 h, or 3) 480 g of VFA (40% acetic acid, 30% propionic acid, and 30% butyric acid) infused intraruminally over 8 h daily beginning at 0630 h. All steers had ad libitum access to prairie hay (5.8% CP; Table 1) fed at 115% of the average voluntary intake over the previous 4 d.

Each experimental period lasted 14 d. The first 9 d were used for adaption to treatments and the last 5 d for sample collection. For the first 7 d of adaption, steers were housed in individual tie-stalls. For the remainder of each period, steers were placed in metabolism crates that allowed for total collection of urine and feces and facilitated intravenous infusion of labeled urea. At 0630 h on d 10 through d 13 of each period, each metabolism crate had a clean bucket containing 900 mL of 10% (wt/wt) H2SO4 placed under the collection funnel to facilitate

complete collection of urine from each steer. The acid maintained the pH of the urine below 3 to prevent ammonia volatilization. Feces were collected into a metal bin lined with plastic from d

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Intake, digestion, and N balance were measured from d 10 through 13. Samples of hay (400 g) were collected on d 9 through 12 and composited within each period to correspond with urine and feces collected from d 10 through 13. Orts were collected just before the daily feeding and orts from d 9 to 12 were composited for each steer. Samples of casein and glucose were collected once each period. Feces and urine collected over a 24-h period were removed each day at 0630 h and sampled. Samples of both feces (5% of total amount collected) and urine (1% of total amount collected) were composited within animal for each period. Two sets of urine samples were collected; one for determination of N balance and another for purine derivative analysis. The urine to be used in purine derivative analysis was diluted 5/1 with 0.05 M H2SO4.

At 4 h after feeding (1030 h) on d 10, blood (10 mL) was collected by jugular venipuncture into heparinized Vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ). Samples were immediately placed in ice and subsequently centrifuged at 1,200 × g for 15 min within 1 h of collection. Plasma was isolated from blood and frozen.

An indwelling ear catheter was placed in each steer on d 10 to allow for infusion of 15

N15N-urea to measure urea kinetics. Sterile saline was infused continuously from the time each catheter was placed until 0630 h on d 11 of each period when infusion of 15N15N-urea solution began through use of a programmable syringe pump (BS-9000 Multi-Phaser, Braintree

Scientific, Inc., Braintree, MA). Label infusion continued through the end of each period. The 15

N15N-urea solution was prepared by combining 3.6 g of 15N15N-urea (99% 15N15N-urea, Medical Isotopes, Inc., Pelham, NH) with 1 L of sterile saline solution (0.9% NaCl). The solution was filter sterilized (0.22 µm filter, Sterivex, Millipore Corporation, Billeric, MA), bottled in glass containers, and stored at 4°C. The infusion rate was 4.16 mL/h.

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Feces (500 g) and urine (100 mL) collected from d 10 were used for measuring background concentrations of 15N. Feces (500 g) and urine (100 mL) collected on d 13 were used to measure the 15N enrichment at plateau (Wickersham et al., 2009).

On d 14 of each period, samples of rumen and duodenal fluid were collected every 4 h for 24 h beginning 2 h after feeding. Whole rumen contents (1.2 L) were collected from each animal to isolate ruminal bacteria. Contents were first strained through 4 layers of cheesecloth, and the liquid portion was collected and analyzed immediately for pH. An 8-mL sample of ruminal fluid was combined with 2 mL 25% (wt/wt) meta-phosphoric acid and frozen for subsequent analysis of VFA. Another 20-mL sample of rumen fluid was mixed with 2 mL of 6 M HCl and frozen for later analysis of ammonia. The remaining fluid and all solids were mixed with 1.0 L of 0.9% (wt/vol) NaCl, blended (NuBlend, Waring Commercial, Torrington, CT) for 1 min and strained through 4 layers of cheesecloth with all liquid collected and frozen for later isolation of bacteria. Bacterial samples collected during each period were composited within animal. Duodenal fluid (300 mL) was collected from each steer concurrent with ruminal samples and was pooled within animal for later analyses.

Laboratory Analyses

The partial DM of hay, ort, and fecal samples were determined by drying in a forced-air oven at 50 °C for 72 h. Duodenal samples were lyophilized. Samples of hay, ort, fecal, and duodenal digesta were ground through a 1-mm screen with a Wiley mill. The DM content of hay, ort, fecal, and duodenal samples as well as casein and glucose was determined by drying for 24 h at 105°C in a forced-air oven and ash content was determined by heating for 8 h in a muffle oven at 450°C.

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Hay, ort, fecal, and duodenal samples were analyzed for NDF (without amylase and without ash correction) and non-sequential ADF using an ANKOM-Fiber Analyzer (ANKOM-Technology, Fairport, NY). To determine acid detergent insoluble ash of fecal and duodenal samples, ANKOM bags containing ADF residues were combusted for 8 h at 450°C in a muffle oven. Protein concentration of casein, hay, ort, duodenal, wet feces, and urine samples was determined through combustion (Nitrogen Analyzer Model FP-2000, Leco Corporation, St. Joseph, MI). Crude protein was calculated as N × 6.25.

Ruminal bacteria were isolated from thawed ruminal samples by centrifuging at 500 × g for 20 min to remove protozoa and feed particles, centrifuging the supernatant at 20,000 × g for 20 min, resuspending the pellet with saline (0.9% NaCl), and centrifuging again at 20,000 × g for 20 min. The bacterial pellet was frozen and lyophilized.

Concentrations of urinary urea (Marsh et al., 1965) and ammonia (Broderick and Kang, 1980) were determined colorimetrically using an AutoAnalyzer (Technicon Analyzer II, Technicon Industrial Systems, Buffalo Grove, IL). Measurement of 15N enrichment in urinary urea was conducted according to Brake (2009). The 15N enrichments of ruminal bacteria, dried feces, and duodenal samples were measured using a stable isotope elemental analyzer

(ThermoFinnigan Delta Plus, Thermo Electron Corporation, Waltham, MA).

Concentrations of allantoin, uric acid, and creatinine were determined in composited (d 10 through 13) urine samples by reverse-phase HPLC as described by Brake (2009).

The method of Vanzant and Cochran (1994) was used to measure VFA in ruminal fluid by GLC. Measurements of ruminal ammonia (Broderick and Kang, 1980), plasma urea-N (PUN; Marsh et al., 1965), plasma creatinine (Chasson et al., 1961), and plasma glucose (Gochman and Schmitz, 1972) were accomplished using an AutoAnalyzer (Technicon Analyzer II). Plasma AA

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were analyzed by GLC using a GC-FID Free Amino Acid Analysis Kit (EZ:faast, Phenomenex, Torrance, CA).

Calculations

Flows to the duodenum were calculated by dividing the fecal output of acid detergent insoluble ash by the acid detergent insoluble ash concentration in duodenal digesta. Microbial N flow to the duodenum was calculated using 2 methods. The first method (“measured”) consisted of multiplying duodenal N flow by the ratio of duodenal 15N enrichment to bacterial 15N

enrichment (Wickersham et al., 2009). The second method (“estimated”) used the methods of Chen and Gomes (1992) to predict microbial N flow from urinary purine derivative excretion. Flow of undegraded intake protein to the duodenum was the difference between total N flow and measured microbial N flow. Microbial N derived from recycled urea was calculated by

multiplying measured bacterial N flow by the ratio of bacterial 15N enrichment

Effects of supplemental energy and protein on forage digestion and urea kinetics in beef cattle